The problem overcome by the present invention is development of an improved non-isotopic, homogeneous microparticle agglutination immunoassay specific for the therapeutic drug phenobarbital without cross-reactivity to other substances in the same class of drugs (barbiturates) based on the kinetic interaction of microparticles (KIMS).
Other non-isotopic immunoassays specific for phenobarbital have used different technologies such as fluorescence polarization immunoassay (FPIA), e.g., Kirkemo et al., U.S. Pat. No. 4,614,823; immunoassays with enzyme-mediated signal generation, e.g., Bogulaski et al., U.S. Pat. No. 4,279,992 (EIA); Flentge et al., U.S. Pat. No. 4,543,412 (EIA); Carrico et al., U.S. Pat. No. 4,255,566 (Flavin-phenobarbital derivatives for EIA); Greenquist, U.S. Pat. No. 4,363,874 (EIA on a membrane); Scholz et al., Recent Dev. Ther. Drug Monit. Clin. Toxicol., 1992, 375-381 (CEDIA); and chemiluminescent immunoassay, e.g., Adamezyk et al., Bioconjugate Chem., 2000, 11, 714-724 (5-position C6-OCONH—R derivatives), while other more esoteric methods have also been used.
There are assays for barbiturates as a class in which phenobarbital has cross-reactivity. (Adamczyk et al., U.S. Pat. No. 6,472,227 [using sec-butyl and cyclopentenyl barbiturate derivatives]; Grote & Hu, U.S. Pat. Nos. 5,099,020 and 5,096,838 [FPIA using 5,5-dialkylbarbiturate derivatives although a phenobarbital derivative is encompassed in the description and claims].
There is also a barbiturate-class microparticle agglutination immunoassay (U.S. Pat. No. 5,618,926) but with a secobarbital-BSA conjugate immobilized on microparticle (as opposed to the phenobarbital analog-aminodextran conjugate in solution format in the present invention). Phenobarbital is one of many with cross-reactivity in the assay.
Buechler (U.S. Pat. No. 5,414,085) describes barbiturate compounds including “5-aryl” substituted substances also synthesized out of the 5-position ending with a thiolactone terminus, although the teaching is directed towards secobarbital derivatives.
Phenobarbital derivatives leashed out of N1 of the barbiturate core, i.e., out of the nitrogen at position 1 rather than out of carbon at position 5 as is the case with compounds of the present invention, are revealed in U.S. Pat. No. 5,284,948, U.S. Pat. No. 5,298,403, EP 517327, and U.S. Pat. No. 5,601,994. See diagram below. The last cited patent also describes an enzyme immunoassay for barbiturates but exemplifies only phenobarbital and its derivatives without indication of crossreactivities of other barbiturates in the assay.

U.S. Pat. No. 5,284,948 and U.S. Pat. No. 5,298,403 also discuss binding of enzyme-labeled phenobarbital derivatives formed from the N1-derivatized substances shown above with antibodies immobilized on beads which are spun down/pelleted in a discrete step, while EP 517327 reveals an enzyme immunoassay using antibodies immobilized on a membrane.